Cancer is a worldwide disease; it was estimated in 2008 [1] that 7.6 million deaths were due to cancer and is expected to rise to 11 million deaths in 2030 [2] . Microbial infections such as viruses, bacteria, and parasites contribute to 17.8% of the global burden of cancer. Cancer cases can be lowered 26.3% in developing countries if these infections are prevented or properly treated. The main causative agents for cancer are Helicobacter pylori (5.5% of all cancer), human papilloma viruses (5.2%), hepatitis B and C viruses (4.9%), Epstein-Barr virus (EBV) (1%), and HIV together with the human herpes virus 8 (0.9%) [3] . Regarding helminthes, Schistosoma haematobium can cause urinary bladder cancer and the flukes Opisthorchis spp. and Clonorchis spp. are associated with cholangiocarcinoma in wide areas of the Far East [4],[5] .

Cancer develops in three stages: initiation, promotion, and progression. The initiation starts by a genotoxic carcinogen, which induces DNA mutation that is irreversible and can persist in the tissues until the initiated cells are exposed to a stimulus or promotion such as irritation and inflammation [6] . Promotion involves the stimulated expansion of a clone of initiated proneoplastic cells, and progression occurs when a clone of early neoplastic cells are transformed into a fully malignant phenotype resulting in uncontrolled growth [7] . Presence of parasites or deposition of their products in tissues induces a chronic inflammation, which is the key element in helminth-induced carcinogenesis [8],[9] . In response to parasites, free radicals are produced from inflammatory cells such as macrophages and eosinophils. These free radicals can oxidize and damage DNA, initiating DNA mutation and leading to genetic instabilities and malignant transformation [9],[10] .

In the present review article, available data on the association of protozoa in neoplastic processes in humans or animals are presented.

Cryptosporidium parvum

Cryptosporidium parvum has been correlated with digestive carcinogenesis. An epidemiologic study in Poland reported a frequency of 12.6% of cryptosporidiosis in patients with colorectal cancer [11] . C. parvum organisms were found to be associated with cystic hyperplasia of the colonic mucosa in experimentally infected nude mice [12] . Another study reported the presence of Cryptosporidium spp. organisms in intestinal polyps of naturally infected sheep [13] . Furthermore, cryptosporidiosis was previously associated with the development of benign tumors in naturally infected vertebrates, having been detected in aural-pharyngeal polyps in naturally infected iguanas using optical or transmission electron microscopy [14] . More recently, the ability of C. parvum to induce gastrointestinal neoplastic changes was established experimentally in severe combined immunodeficient mice [15],[16],[17] . Adenomas with low-grade or high-grade intraepithelial, intramucosal, or invasive neoplasia associated with numerous C. parvum life stages were detected in different areas of the digestive tract including stomach, duodenum, and ileocecal region. A study carried out in 2012 showed that C. parvum induced digestive adenocarcinoma by infection with low doses of Cryptosporidium spp., even with only one oocyst. In mice inoculated with low doses, neoplastic lesions were detected as early as 45 days postinfection in the stomach and ileocecal region, which could evolve in an invasive adenocarcinoma [18] . In the same year, a study was carried out to report and characterize a C. parvum strain isolated from a patient who developed fulminant cryptosporidiosis. The strain isolated from the patient's stools, identified as C. parvum II2A15G2R1 (subtype linked to zoonotic exposure), was inoculated into severe combined immunodeficient mice. In this host, this virulent C. parvum isolate induced not only severe infection, but also invasive gastrointestinal and biliary adenocarcinoma. The observed progress of the adenocarcinomata through all layers of the digestive tract to the subserosa and its spread by blood vessels confirmed the invasive nature of the neoplastic process. These results indicate for the first time that a human-derived C. parvum isolate is able to induce digestive cancer. This study is of special interest considering the exposure of a large number of humans and animals to this waterborne protozoan, which proved highly tumorigenic when inoculated in a rodent model [19] . In another study carried out in Maryland, USA, the investigators analyzed 320 incident colorectal cancer cases that occurred among 471,909 patients with AIDS. Colorectal cancer risk was elevated following cryptosporidiosis [20] . Few data about the mechanism of C. parvum-induced neoplasia are available. Modulation of apoptotic pathways was investigated by microarray analysis in an in-vitro model using human ileocecal HCT8 cells. Genome-wide expression profiling revealed high proportion of apoptosis genes regulated during C. parvum infection. Analysis of the apoptosis gene transcript profile suggested a biphasic control of host-cell apoptosis. BCL2 and c-Myc genes showed an altered expression during C. parvum infection in HCT8 cells [21] . In 2014, an experimental study on mice showed that metabolic pathways are potentially involved in the development of C. parvum-induced ileocecal oncogenesis. They detected alterations in Apc and β-catenin expression in infected mice, implicating the Wnt signaling pathways in C. parvum-induced neoplasia. Furthermore, immunohistochemical analysis revealed abnormal localization of β-catenin at a basolateral position, and transmission electron microscopy revealed dilation of intercellular spaces with development of lateral membrane extensions at the level of adherens junctions. These data indicate that C. parvum is able to modulate host cytoskeleton activities and several host-cell biological processes [22] .

Toxoplasma gondii

The first recorded work on the relationship of central nervous system neoplasias with Toxoplasma gondii infection was carried out in 1967. The authors found an association between Toxoplasma spp. infection and central nervous system neoplasms, specifically astrocytomas [23] . Later on, investigators revealed that T. gondii could cause gliomas in experimental animals [24] . Other studies carried out by Ryan et al. [25] showed that antibody positivity to Toxoplasma spp. is associated with meningioma rather than glioma. An epidemiological study analyzed data from 37 countries for the incidences of adult brain cancers and Toxoplasma spp. infection and recorded an associated 1.8-fold increase in the risk for brain cancers across the range of prevalence of Toxoplasma spp. infection [26] . In France, recently conducted research showed that mortality rates due to brain cancer correlated positively with the local seroprevalence of Toxoplasma spp., particularly in men who are 55 years of age or older [27] . Thomas et al.[26] predicted that T. gondii could increase the risk for brain cancer because it is a long-lived parasite that encysts in the brain, where it provokes inflammation and inhibits apoptosis. In contrast, it was reported that cells infected with T. gondii are resistant to multiple inducers of apoptosis, including Fas-dependent and Fas-independent cytotoxic T lymphocyte-mediated cytotoxicity, interleukin-2 (IL-2) deprivation, g irradiation, UV irradiation, and the calcium ionophore beauvericin. The report hypothesized that inhibition of such a broad array of apoptosis inducers suggests that a mechanism common to many, or perhaps all, apoptotic pathways is involved and concluded that the inhibitory activity requires live intracellular parasites and ongoing protein synthesis [28] . From China, came the first report to describe pituitary adenoma in association with T. gondii infection in two patients. Radiological examination revealed tumors in the sellar region. Microscopically, the tumors consisted of small homogeneous polygonal or round cells. Toxoplasma spp. cysts were found among the tumor cells, a finding confirmed by T. gondii-specific antibody immunohistochemistry. The association between pituitary adenoma and Toxoplasma raised the possibility that T. gondii might be involved in the development of certain cases of pituitary adenoma [29] . When an experimental study showed that exogenous prolactin has antiparasitic effects, it was postulated that overstimulation of pituitary gland to fight the parasitic infection may lead to adenoma formation [30] .

A more recent study showed that Toxoplasma spp. is capable of manipulating host micro RNAs (miRNAs), which play a central role in post-transcriptional regulation of gene expression [31] . The researchers hypothesized that Toxoplasma spp. promotes brain carcinogenesis by altering the host miRNAome using parasitic proteins and/or miRNAs.

Other researchers studied the relationship between toxoplasmosis and different body neoplasms. Rocchi et al.[32] perceived that the incidence of high titer of specific T. gondii antibodies was greater in patients with Hodgkin's disease in comparison with controls. Researchers from USA and Switzerland reported 10 cases of primary intraocular lymphomas that were analyzed using microdissection and PCR. T. gondii DNA was detected except in normal cells of two cases that had clinically resembled ocular toxoplasmosis. The results led the researchers to speculate that T. gondii may play a role in some forms of primary intraocular B-cell lymphoma [33] . In Nepal, seroprevalence of Toxoplasma spp. infections in 272 patients with ocular diseases (uveitis and retinochoroiditis), malignancy (including leukemia), women with bad obstetric history as well as patients with fever, lymphadenitis, and encephalitis has been studied. Patients with ocular malignancy had the highest positive rate of toxoplasmosis (68.7%) followed by the other groups [34] . In Turkey, 108 patients with neoplasia and an equal number of healthy controls were studied for the presence of anti-T. gondii antibodies. Specific immunoglobulin G (IgG) antibodies were detected in 63.0% of patients and in 19.4% of controls, which was a statistically significant difference. In addition, specific IgM antibodies were detected in 6.5% patients and 0.9% controls [35] . In addition, T. gondii antibodies determined in 267 cancer patients had higher positivity rates of IgG compared with controls. The positivity rates of T. gondii IgG in the nasopharyngeal carcinoma and rectal cancer groups were significantly higher than in the other cancer groups, but the differences in IgM positivity rates were not significant [36] .

Trichomonas vaginalis

The possible association between Trichomonas vaginalis and cervical neoplasia has been studied since the 1950s. A study carried out on 1966 patients showed that the frequency of cervical carcinoma in association with T. vaginalis infection in USA was three times as high as for individuals with negative T. vaginalis smears [37] . The same results were obtained in Canada [38] . In Singapore, it was shown that T. vaginalis antibodies were detected in sera from 41.3% of patients with invasive cervical cancer compared with only 5% of female controls. The recorded significantly increased relative risk = 3-42 (95% confidence interval = 1.73-6.78), proved comparable with the relative risks derived in seroepidemiological studies of human papilloma virus, suggesting that T. vaginalis may be even more closely associated with invasive cervical cancer than previously realized [39] . However, in Chinese women, 4-5% of cervical cancer cases were attributed to T. vaginalis infection [40] . In a large cohort study conducted in Finland in 2000, T. vaginalis was associated with a high relative risk of cervical cancer 6.4 [41] . In Egypt, Sayed El-Ahl et al. [42] detected antibodies to T. vaginalis in sera from 18.75% of patients with cervical cancer compared with 5% of controls.

Additional proof of the association between cervical neoplasia and trichomoniasis comes from serological and/or histopathological studies. A study carried out in Egypt in 2007 showed that, of 150 women with vaginitis, 46 were specifically diagnosed as trichomoniasis. In Pap smears, 10 revealed atypical squamous cells of undetermined significance, two smears showed atypical high-grade squamous cells, eight smears contained squamous intraepithelial low-grade lesions in the form of mild dysplasia, and four smears showed high-grade squamous intraepithelial lesions in the form of moderate-to-severe dysplasia and carcinoma in-situ[43] . Moreover, a study carried out to analyze the modifications at the junctional complex level of Caco-2 cells after interaction with two isolates of T. vaginalis confirmed that T. vaginalis adheres to the epithelial cell causing alterations in the junctional complex, in the form of a decrease in transepithelial electrical resistance, alteration in the pattern of junctional complex proteins distribution as observed for E-cadherin, occludin, and ZO-1, and enlargement of the spaces between epithelial cells. These effects were dependent on the degree of parasite virulence of the isolate, the iron concentration in the culture medium, and the expression of adhesin proteins on the parasite surface [44] . In Malaysia, it was confirmed that there are phenotypic 'variant' forms of T. vaginalis trophozoites isolated from patients suffering from cervical neoplasia condition, where the trophozoites isolated from the neoplasia proliferated at a higher rate and contained large numbers of hydrogenosomes and vacuoles implying that these are virulent forms that can aggravate or exacerbate cervical neoplasia [45] . At the same time, a study confirmed that a pseudocystic stage form found in cervical neoplasia demonstrated different biochemical, surface, and ultrastructural properties, despite its uniform appearance under light microscopy of being rounded without a true cyst wall and showing no motility. The study provided evidence that phenotypic variant forms of pseudocysts do exist and possibly play a role in exacerbating cervical cancer [46] .

The first study to investigate associations between T. vaginalis serology and prostate cancer was conducted in 2006. A large, nested case-control study on male health professionals with incident prostate cancer, carried out on 691 USA cases and an equal number of controls, found 13% cases and 9% of controls seropositive for trichomoniasis. It was reported that serologic evidence of a history of trichomoniasis was positively associated with prostate cancer [47] . In addition, another seropositive status was found to be associated with statistically significantly increase in risk for metastatic prostatic cancer or prostate cancer-specific death [48] . It was also shown that T. vaginalis mediated intraprostatic inflammation and cell damage. In Taiwan, a study carried out to investigate effect of modification by toll-like receptor 4 (TLR4) variation on this association hypothesized that TLR4 variation might serve as a marker of antitrichomonad immune response, because T. vaginalis has been shown to elicit inflammation through this receptor. TLR4 variation appeared to influence the association between T. vaginalis serological status and prostate cancer risk, consistent with the hypothesis that inflammation plays a role in this association [49] .

Blastocystis hominis

Although Blastocystis hominis is one of the most common parasites found in any stool survey, there has been limited research on its association with colorectal cancer. RT-PCR revealed that colon cancer cells express significantly higher levels of IL-6 and IL-8 in the presence of Blastocystis spp. antigen [50] . Previously, IL-6 was associated with proliferation of colon carcinoma in a number of reports [51],[52],[53] . A study carried out in 2010 concluded that the solubilized antigen of Blastocystis spp. facilitates the in vitro proliferation of human colorectal carcinoma cells (HCT116). The gene expressions of cytokines namely IL-6, IL-8, tumor necrosis factor α, interferon-g (IFN-g), nuclear factor k light-chain (NF-kb) enhancer of activated B-cells (a gene transcription factor), and proapoptotic genes namely protein p53 and cathepsin B were studied. Antigen from B. hominis, at a certain concentration, could facilitate the growth of HCT116 cells while having the ability to down regulate immune cell responses [54] . As reported, a significant proliferation of these cells was observed when exposed to 1.0 μg/ml solubilized antigen isolated from subtype 3 Blastocystis spp. There was up regulation of Th2 cytokines especially transforming growth factor β in subtype 3-treated cancer cells. In addition, there was a significantly higher up regulation of cathepsin B, which led to the postulation that it may enhance the exacerbation of existing colon cancer cells by weakening the cellular immune response. The dysregulation of INF-g and p53 expression also suggested Blastocystis spp. as a proponent of carcinogenesis [50],[54] . Therefore, it is very likely for subtype 3 Blastocystis spp. to have higher pathogenic potential as it caused an increased propagation of cancer cells and substantial amount of inflammatory reaction compared with other subtypes [50] . This confirmed a study carried out in Germany, which speculated that Blastocystis spp. has the ability to down regulate the host immune response at the beginning stage of colorectal cancer to improve its survival [55] . In addition, antigen isolated from a symptomatic individual is more pathogenic as compared with asymptomatic isolates as it caused a more extensive inflammatory reaction as well as more enhanced proliferation of cancer cells [56] . It was concluded that there is a vital need to screen colorectal cancer patients for B. hominis infection as it possesses the ability to enhance tumor growth [54] . Oxidative stress, which has been implicated as an important pathogenic factor in the pathophysiology of various life-threatening diseases such as cancer, was observed to cause oxidative damage in rats inoculated with human-derived Blastocystis spp. isolate [57] .

Plasmodium falciparum

Endemic Burkitt lymphoma is linked to Plasmodium falciparum infection. In East and Southern Africa during the early 1960s, Burkitt [58] assessed the geographical distribution and the incidence of a sarcomous lymphoma. Burkitt found its presence to be correlated with the same temperature and rainfall zones as malaria. In 1982, malaria and EBV were identified as cofactors in the pathogenesis of endemic Burkitt lymphoma [59] , which is estimated to account for 70% of cancers among children in equatorial Africa [60],[61] . EBV infection is followed by life-long infection in memory B-lymphocytes [62],[63] . P. falciparum induces polyclonal B-cell expansion and lytic EBV reactivation, thus increasing the number of latently infected B-cells [64] . Repeated P. falciparum malaria infections could lead to exhaustion or hyporesponsiveness of EBV latent or lytic antigen CD8+T-cells, thus increasing the chance for this EBV-associated malignancy to arise [65] . Moreover, recent evidence indicates that P. falciparum exacerbates the oncogenic effects of EBV by activating EBV replication [64] .

In the USA, a study correlated age-specific patterns of 2602 cases of African Burkitt lymphoma (60% male, mean ± SD age = 7.1 ± 2.9 years) from Uganda, Ghana, and Tanzania with malaria biomarkers published from these countries. Age-specific patterns of this disease and mean multiplicity of P. falciparum malaria parasites, defined as the average number of distinct genotypes per positive blood sample based on the merozoite surface protein-2, were correlated and both peaked between 5 and 9 years. This pattern, which was strong and consistent across regions, contrasted parasite prevalence that peaked at 2 years and decreased slightly and geometric mean parasite density that peaked between 2 and 3 years and decreased sharply. These findings suggest that concurrent infection with multiple malaria genotypes may be related to onset of African Burkitt lymphoma [66] .

In 2010, another study showed a relationship between malaria in the USA and brain tumor incidence, seen in data on malaria outbreaks in 2004 from the Centers for Disease Control and Prevention [67] and reports of brain tumor incidence by state from 19 USA states [68] . The association of malaria with brain tumors was significant. The association between malaria and cancer mortality can be possibly explained by the well-established ability of Plasmodium spp. to induce suppression of the immune system leading to an increased susceptibility to many secondary infections. In addition, in 2010 a second explanation was proposed indicating that the Anopheles spp. mosquito, the vector of malaria, transmits an obscure virus that initially causes only a mild transitory illness but much later predisposes to cancer [69] .

Conclusion

There are extensive epidemiological evidences that some parasites can be a factor of various malignant tumors, but it is difficult to assess the cause of this relationship. More research is needed to find links between clinical, epidemiological data, molecular factors, parasites, and cancer development.

Acknowledgements

The authors would like to thank Professor Dr. Sherif M. Abaza, Professor of Parasitology, Faculty of Medicine, Suez Canal University for his valuable revision of the article.